Research Article

Received: 10 June 2014, Revised: 15 September 2014, Accepted: 29 September 2014 Published online in Wiley Online Library: 25 November 2014

(wileyonlinelibrary.com) DOI 10.1002/ffj.3225

Preparation and biological activity of

nanocapsulated Glycyrrhiza glabra L. var.
glabra
Akbar Esmaeili* and Roghayeh Rafiee
ABSTRACT: Glycyrrhiza glabra is a medicinal plant found in Asia, the Mediterranean and parts of southern Europe. It used for the
treatment of upper respiratory tract ailments including coughs, hoarseness, sore throat and bronchitis. In this study, natural
polymers, chitosan and alginate were used for preparing nanocapsules containing turmeric oil and a methanol extract of G.
glabra. The antimicrobial activity of G. glabra as an extract and in a nanocapsule was investigated. In the preparation of
nanocapsules, a three-step procedure involving emulsification, cross-linking with calcium chloride and solvent removal was
used. Morphological characteristics of the nanocapsules were investigated by particle size using transmission electron micros-
copy. The results showed that turmeric oil-to-extract ratio, surfactant and molecular weight of chitosan all affect the size of
the nanocapsule produced. An oil-to-extract ratio of 10:10 mg/ml, Tween 80 and low molecular weight chitosan resulted in the
optimal (smallest) nanoparticle size. Copyright © 2014 John Wiley & Sons, Ltd.

Keywords: Nanocapsule; chitosan; alginate; Glycyrrhiza glabra; turmeric oil

Introduction and texture are important factors.[6] Nanoencapsulation has been

investigated as a means of protecting palmitate structures against
The volatile oils extracted from plants have wide industrial applica- photodegradation by ultraviolet-visible spectrophotometry
tions for such commercial products as drugs, detergents, perfumes (UV-Vis) radiation.[7] Esmaeili and Niknam[8] have recently improved
and herbal teas.[1] Herbal therapies have been used in the the nanocapsulation of Elaeagnus angustifolia, which when used as
treatment of illnesses since ancient times. In recent years, the a drug has hypotensive effects and also acts as a direct and mild
negligible adverse effects of these formulations have increased heart tonic. In their study, Esmaeili and Niknam[8] used olive oil as
their popularity worldwide. Glycyrrhiza glabra L. var. glabra, or a ‘carrier’ for nanocapsulation. Turmeric oil has also been used for
licorice, is a native plant of southeast Europe, southwest Asia and this purpose. Turmeric oil is widely used in pharmaceutical and
Iran. This sweet, moist, soothing herb has anti-inflammatory and cosmetic applications because of its antibacterial, antifungal, antiox-
expectorant properties and can control coughing, as well as idant and insect-repellent properties. The potential of G. glabra
displaying hormonal effects.[2] The roots and rhizomes of G. glabra nanocapsules as a novel method for drug delivery as well as
are used extensively in herbal medicines for their emollient, functional ingredients in foods indicates a need for studies on the
anti-inflammatory, antiviral, anti-allergenic, antioxidant, biological activity and conditions that influence the characteristics
gastroprotective and anticancerous properties.[3] In this study, of G. glabra nanocapsules.[9]
the biological activity of G. glabra extract and G. glabra
nanocapsules were investigated.
The biocompatibility, biodegradability and non-toxicity of Materials and Methods
natural polymers have led to an interest in their use as drug-
delivery systems for humans. The most well-known of these Chemicals
polymers is chitosan: one of the natural polymers used in this Chitosan (low and medium molecular weight), sodium alginate,
study. The most important derivative of chitin is chitosan, Tween 80, Tween 60, polyvinyl alcohol (PVA) and calcium chloride
obtained by partial deacetylation of chitin in the solid state were purchased from Fluka Co, (Buchs, Switzerland). Di (phenyl)-(2,
under alkaline conditions (concentrated NaOH) or by enzymatic 4, 6-trinitrophenyl) iminoazanium (DPPH), vitamin C (ascorbic acid)
hydrolysis in the presence of a chitin deacetylase.[4] and Folin–Ciocalteu’s reagent were supplied from Sigma-Aldrich
Another natural polymer used in this study was sodium alginate, (St Louis, MO, USA). Potassium ferricyanide [K3Fe(CN)6], trichloro-
which is commonly used in pharmaceutical and biomedical appli- acetic acid and phosphate buffer (2.5 ml, 0.2 M, pH 6.6), purchased
cations. Sodium alginate consists mainly of the sodium salt of
alginic acid, which is a mixture of polyuronic acids composed of
residues of d-mannuronic acid and l-guluronic acid obtained *Correspondence to: A. Esmaeili, Department of Chemical Engineering, North
mainly from brown algae.[5] The nanoencapsulation of medicinal Tehran Branch, Islamic Azad University, PO Box 19585/936, Tehran, Iran.
E-mail: [email protected]
plant extracts has many applications in drug manufacturing.
Because of their transparency and small droplet size, nanocapsules Department of Chemical Engineering, North Tehran Branch, Islamic Azad
also have applications in formulation of foodstuffs in which colour University, Tehran, Iran
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Flavour Fragr. J. 2015, 30, 113–119 Copyright © 2014 John Wiley & Sons, Ltd.
 A. Esmaeili and R. Rafiee

from Merck Co. (Darmstadt, Germany), were used in this study.

Turmeric powder was purchased from Navidkaran Co. in Iran.

Plant Extraction
Dried, finely powdered aerial parts of G. glabra (83.25 g) were
extracted with methanol (125 ml) in a Soxhlet extractor for 2 days.
After filtration of the extract, a rotary evaporator was used to
remove the methanol. The crude residue (28.30 g) remaining was
washed with n-hexane (125 ml). Following this, the residue was
dissolved in hot deionized water (85 ml) and extracted first with
ethyl acetate (85 ml thrice) and then with butanol. The material
remaining after evaporating all the solvent (rotary evaporator)
was used in this study. In this method, 50 g of turmeric powder
was added to 500 ml distilled water in a flask, the flask attached
to a Clevenger distillation head and then heated (refluxed) for
about 2 h. The essential oil was collected, dried under anhydrous
sodium sulfate and stored at 4 °C until used.

Synthesis of Nanocapsules under Varying Conditions

Chitosan extracted from shrimp-shell waste
First, shrimp shells were separated from waste materials, dried
and then powdered. To deproteinize the skin powder, sodium
hydroxide solution (2%) was added with 1:30 (v/w) ratio (1 g of Scheme 1. The nanocapsulation manufacturing process
skin powder and 30 ml of sodium hydroxide) and the product
was incubated for 2 h at 90 °C. The precipitate was washed with Antioxidant Activity under different methods
distilled water and then 10% acetic acid (in distilled water) at
1:40 (v/w) ratio (1 mg of precipitate and 40 ml of 10% acetic acid) In this study the antioxidant properties of G. glabra extract and
was added to the precipitate in order to extract the chitosan. G. glabra nanocapsules were determined without using an acid
Then, the product was incubated for 6 h at 60 °C. In order to reagent. The nanocapsules are covered by polymer as using acid
recover the supernatant phase containing dissolved chitosan in can destroy the capsules.
acetic acid, the solution was centrifuged at 4000 rpm for 1,1-Diphenyl-2-picrylhydrazyl radical
15 min. Finally, for separation the chitosan, 4 M NaOH was added
to the solution until the pH reached 9. The chitosan was precip- In order to determine the free radical-scavenging activity of the
itated from the solution and placed in desiccators for 48 h to be free G. glabra extract and G. glabra nanocapsules, a 1,1-diphenyl-
dried. 2-picrylhydrazyl (DPPH) assay was used. Keeping the volume con-
stant, different concentrations of G. glabra extract and G. glabra
nanocapsules (50, 100, 200, 400, 800 μg/ml) were added to a
Preparation of chitosan–alginate nanocapsules methanol solution of DPPH (100 μM). The solution was left to sit
A method described by De and Robinson was used to prepare for 15 min at room temperature and then adsorbance was
chitosan–alginate nanocapsules.[10] Briefly, an o/w emulsion was determined at 517 nm.[11]
made by slowly dropping 0.6 ml of extract (containing 10 mg/ml Hydrogen peroxide
methanolic turmeric oil solution and 10 mg/ml methanolic extract
of G. glabra – which is the organic phase) into 20 ml of aqueous Na Using a method previously described in a study of Stoechospermum
alginate (0.6 mg/ml) containing 1% (w/v) Tween 80 (which is the wa- marginatum, we evaluated the hydrogen peroxide scavenging of
ter phase). The mixture was sonicated for 20 min and then combined G. glabra extract and G. glabra nanocapsules.[11] A solution of hydro-
with 4 ml of 0.67 mg/ml CaCl2 solution and stirred for 30 min. This gen peroxide (40 mm) was prepared in phosphate buffer (pH 7.4). In
nanocapsule suspension was then combined with 4 ml of chitosan two separate experiments (replicates), samples (0.1, 0.2, 0.4, 0.8 and
solution (0–0.6 mg/ml in 1% [v/v] acetic acid) and stirred for 30 min. 1 mg/ml) were mixed with distilled water and then added to a
Rotary evaporation was used (40 °C for 20 min) to remove the solvent, hydrogen peroxide solution (0.6 ml, 40 mm). Ten minutes were
resulting in a chitosan–alginate nanocapsule suspension loaded with allowed to pass and we then determined the absorbance (i.e. hydro-
turmeric-oil–licorice extract. The nanocapsulation manufacturing gen peroxide) at 230 nm, which was then compared against a blank
process described above is shown in Scheme 1. solution containing phosphate buffer without hydrogen peroxide.
The following formula was used to calculate the percentage of
hydrogen peroxide scavenging by the extracts and a standard
Variation of parameters in the formulation compound:
The chitosan molecular weight, surfactant type and concentration,
scavenged H2 O2 % ¼ ½ðAo  A1 Þ=Ao 100
and the oil-to-extract concentration ratios were varied to determine
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their effect on particle size (Table 1). where Ao was the absorbance of the control and A1 was the

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Characterization of nanocapsules containing G. glabra

Table 1. Results for average size of particle for variable parameters

Sample Turmeric G.g-E Type of Chitosan Surfactant Surfactant Average size of

oil/G.g-E ratio (mg) Chitosan (mg) type (mg) particle (nm)
1 1.1 0.1 LM 0.02 PVA + T 80 0.06 + 0.03 152
2 1.1 0.1 LM 0.04 PVA + T 80 0.06 + 0.03 205
3 1.1 0.1 ES 0.04 T 80 0.02 90.9
4 1.1 0.1 LM 0.02 T 80 0.02 89.5
5 1.1 0.1 LM 0.02 T 80 0.02 71.9
6 1.1 0.2 LM 0.06 T 80 0.07 251
7 1.1 0.2 LM 0.06 T 80 0.02 181
8 1.1 0.2 MM 0.06 T 80 0.02 122
9 1.1 0.2 LM 0.06 T 80 0.02 119
10 1.1 0.2 LM 0.06 T 80 0.03 120
11 1.1 0.2 LM 0.06 T 80 0.05 54.3
12 1.1 0.2 LM 0.06 T 80 0.02 57.7
13 1.1 0.2 LM 0.06 T 80 0.02 181
14 1.3 0.15 LM 0.06 T 80 0.02 111
15 3.1 0.05 LM 0.06 T 80 0.02 103
G.g-E, Glycyrrhiza glabra extract; LM, low molecular weight; MM, medium molecular weight; ES, extract from shrimp; PVA, polyvinyl
alcohol; T 80, Tween 80.

absorbance in the presence of the sample of extract and standard The methanol fraction was filtered through a paper filter and
compound. then the methanol was removed in a rotary evaporator at 60 °C.
The antimicrobial activity of the sample extracts was evaluated
Total phenolics
against Staphylococcus aureus (PTCC 1169), Enterococcus faecalis
Phenolic compounds were measured according to the methods (PTCC 1447), Acinetobacter baumannii (ATCC 17978), Bacillus cereus
in which Folin–Ciocalteu is used as the reagent and gallic acid as (PTCC 1023), Pseudomonas aeruginosa (ATCC 27853) and Klebsiella
the standard.[11] We well-mixed 0.5 ml of G. glabra extract pneumonia (PTCC 1053) using the diffusion method (Table 2).
solution with different concentrations (50, 100, 200, 400 and Extract samples were incubated for 24 h in cultures of each micro-
800 μg/ml) and 50 ml of methanol and 1 ml Folin–Ciocalteu organism. Following this, the minimum inhibitory concentration of
reagent. After 5 min, 3 ml of 2% sodium carbonate solution were each extract was evaluated by measuring the diameter of the
added and the mixture was continuously shaken for 2 h. Then, non-growth zone. This study is based on experiments conducted
the sample absorbance was measured at a wavelength of according to the NCCLS 2OO4 standards and instructions and
765 nm. The same procedure we used again for 0.1, 0.2, 0.3, does not require statistical analysis.[11] The above method was
0.4, 0.5 and 0.6 mg/ml of all standard solutions of Gallic acid to carried out for nanocapsules and results were compared.
prepare the standard curve. The above procedure was repeated
for nanocapsules separately.
Statistical Analysis
Antibacterial Activity
All statistical analysis was carried out using SPSS 12 for Windows.
In this study the antimicrobial activities of G. glabra extract and The statistical significance of differences among values was
G. glabra nanocapsules were studied. The G. glabra extract powder assessed using the one-way ANOVA test. Error bars are indicated
was mixed with methanol and the mixture was refluxed for 2 days. wherever necessary.

Table 2. Antibacterial activity of G. glabra nanocapsule and G. glabra extract based on dilution methoda

Bacterial species Gram NGZD MIC (μg)

+/
G. glabra nanocapsule G. glabra extract G. glabra nanocapsule G. glabra-extract
Staphylococcus aureus (PTCC 1169) + 1 1.5 180 160
Enterococcus fecalis (PTCC 1447) + 2 1.5 180 160
Bacilus cereus (PTCC 1023) + 0.5 0.7 250 210
Acinetobacter baumannii (ATCC 17978)  1.2 1.5 150 100
Pseudomonas aeruginosa (ATCC 27853)  1.6 1.4 160 140
Klebsiella pneumonia (PTCC 1053)  2 1.5 150 140
MIC, minimum inhibitory concentration; NGZD, non-growth zone diameter.
a
Values are the mean diameter of inhibitory zones (cm).
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 A. Esmaeili and R. Rafiee

Results and Discussion Oil-to-Extract Ratio

The optimal ratio of oil to extract for nanocapsule preparation
Molecular Weight of Chitosan
was determined by examining ratios of 0:20, 5:5, 10:10, 15:5,
The average size of the chitosan–alginate nanocapsules had a 5:15 and 20:20 mg/ml (turmeric oil to G. glabra extract). As
close relationship with the molecular weight of the chitosan expected, nanocapsule size strongly correlated with the amount
(F-value =42.453). Confirming a study by Lu et al.,[12] the size of of added extract. The F-value is non-negative, with a value of
the nanocapsules increased when higher molecular weight chitosan 21.640 (Figure 1). Increasing turmeric oil and/or G. glabra extract
was used (this is assumed to be due to its higher viscosity). As the formed larger nanocapsules, for example, oil/extract ratios of
goal was to make smaller particle size microcapsules, the lower 20:20 and 5:15 mg/ml produced nanocapsules that were
molecular weight chitosan was chosen as the best cationic biopoly- 108 nm and 89 nm, respectively. On the other hand, results
mer for our purposes. showed that if the amount of oil was less than the amount
Confirming a study by Prego et al.,[13] attaching a polymer to of extract, the resulting particle shape was irregular. Based
the surface of the oil and extract core (e.g. alginate) led to an on the results above, the mass ratio of oil to extract of
increase in the size of the particle. Increases in the size of the 10:10 mg/ml was chosen as the best ratio for creating stable
nanocapsule show that chitosan is located on the alginate polymer nanocapsules. The morphology of capsules produced at this
surface; this process must have occurred as a result of electrostatic ratio was examined using transmission electron microscopy
interactions between the negative part of the carboxylate groups in (TEM; Philips EM208 keV, Eindhoven, The Netherlands), which
alginate and the positive part of calcium ions and protonated amino showed that the capsules were round and that their size was
groups on chitosan, and proved useful for stabilizing the oil core. 50 nm (Figure 2).

Figure 1. The effect of different oil/extract mass ratios on nanocapsule size

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Figure 2. A TEM of prepared nanocapsules

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Characterization of nanocapsules containing G. glabra

Concentration and Type of Chitosan presence of PVA, and smaller particles when Tween 80 and 60 were
used. The determined F-value for the effect of surfactants was
As noted earlier, the molecular weight of chitosan has a substantial
42.453. The effect of different amounts of surfactant on nanocapsule
effect on the size of the nanocapsules. Furthermore, the concentra-
size was also investigated (Figure 5), with a calculated F-value equiva-
tion ratio between alginate and chitosan has an important role in
lent to 5.534. Findings indicated that the effect of surfactants on par-
determining the size of nanocapsules. In this section, the effect of
ticles size is highly significant (Figure 5). (Data are summarized in
different concentrations of chitosan (0.1 to 0.6 mg/ml) on the size
Table 1).
of nanocapsules is reported (Figure 3). The F-value (16.953) is the
statistic calculated by ANOVA. The results show that nanocapsule
size has a direct correlation with the concentration of chitosan, that Antioxidant Activity
is, the size of nanocapsules increased with higher amounts of Amount of DPPH
chitosan. As pointed out by Sarmento et al.,[14] increasing the
Glycyrrhiza glabra extract demonstrated a significant antioxidant
concentration of chitosan causes a strong interaction between the
effect. The hydrogen donating ability is thought to be the main
polymers, which leads to the forming of polyelectrolyte complexes
reason for the effect of antioxidants on DPPH radical scavenging.
with greater stability. Due to this phenomenon, a mass ratio of 0.1:1
DPPH is a stable free radical that accepts an electron or hydro-
of chitosan/alginate was selected to produce stable nanocapsules.
gen radical to become a stable diamagnetic molecule.
Another factor studied was the ability to make nanocapsules from
Glycyrrhiza glabra extract and G. glabra nanocapsules were
chitosan extracted from shrimp waste (i.e. skin). After extraction of
found to be successful in reducing the stable violet DPPH radical
chitosan from shrimp waste, Fourier transform infrared spectros-
to the yellow diphenylpicrylhydrazyl with a median inhibition
copy (FTIR) and gas chromatography–mass spectrometry (GC–MS)
concentration (IC50) value. The antioxidant molecule provides a
of the samples were compared with the sample that had been
hydrogen atom, or donates an electron, to DPPH.[15] Scavenging
purchased. The FTIR and GC–MS spectra were compared with each
stable DPPH radicals is a method commonly used to evaluate
other and the results showed that the spectra are well matched with
antioxidant activities in a relatively short amount of time.[11] The de-
each other (Figure 4), suggesting that shrimp skin chitin can be used
crease in the absorbance of DPPH radical at 517 nm induced by an-
to make microcapsules.
tioxidants points to their reducing abilities. The concentrations of G.
glabra extract and G. glabra nanocapsules required for scavenging
50% of the free radicals (IC50) was 197 and 185 ± 2.1 μg/ml,
Surfactant
respectively. Trigonella monantha, Salvia glutinosa, Tanacetum
To study the effect of surfactants on the nanocapsule size, different pinnatum and Stoechospermum marginatum have been reported
types of surfactant, that is, Tween 60, 80 and a mixture of Tween 80 to exhibit antioxidant activity in the DPPH test system.[11,16,17] A
and PVA, were used. The results indicated larger particles in the comparison between our research and other studies showed a

117

Figure 3. The effect of amount of chitosan on nanocapsule size

Flavour Fragr. J. 2015, 30, 113–119 Copyright © 2014 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/ffj
 A. Esmaeili and R. Rafiee

Figure 4. The FTIR of (a) clutosan extracted from shrimp waste and (b) purchased chitosan

Figure 5. The effect of different amounts of surfactant Tween 80 on nanocapsule size

difference in IC50 values that can be attributed to the abundance of equivalent referenced against a standard curve (y =0.0089x – 0.0085,
flavonoids in G. glabra: these compounds exhibit high antioxidant R2 = 0.9676). Our findings demonstrate that the metabolic extract of
activity. The G. glabra nanocapsules showed lower antioxidant G. glabra contains a substantial amount of phenolics. However, the
activity in DPPH assay than the G. glabra extract. The reason for this amount of phenolic material cannot be absolutely determined by
is that the nanocapsules are coated with polymer, which does not the Folin–Ciocalteu method, and dependent on structure, different
have a direct effect on the free radical. phenolic compounds exhibit different antioxidant activity. Glycyrrhiza
glabra extracts contain a mixture of phenolic compounds, which have
Total phenol content different antioxidant capacities.
Phenols and polyphenolic compounds, such as flavonoids, are
Hydrogen peroxide scavenging
produced by plants and microorganisms and exhibit significant
antioxidant activities.[18,19] As noted earlier, Folin–Ciocalteu was used Our findings showed that IC50 values for G. glabra nanocapsules
118

as a reagent and therefore results are reported in terms of gallic acid and G. glabra extract were 293 ± 2.7 and 195 ± 1.75 μg/ml,

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Characterization of nanocapsules containing G. glabra

respectively. For reference, the IC50 value for ascorbic acid was small nanocapsules. Among the investigated surfactants, Tween
495 ± 1.8 μg/ml. Hydrogen peroxide can cause cytotoxicity by 80 produced the smallest particles at lower concentrations. The pre-
maximizing the number of hydroxyl radicals in the cell. Hydrogen ferred ratio of turmeric oil to G. glabra extract was determined to be
peroxide itself is not very reactive, yet it can be toxic to cells 10:10 mg/ml, based on the effect on both particle size and particle
because it may give rise to hydroxyl radicals within the cells.[20] shape.
Thus, removing H2O2 is very important for antioxidant defence in
cell or food systems. The expected biological activity observed
for G. glabra would be quite different if we used an acid to open References
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